Identification of Quantitative Trait Loci (QTL) for Sucrose and Protein Content in Soybean Seed.

Protein and sugar content are important seed quality traits in soybean because they improve the value and sustainability of soy food and feed products. Thus, identifying Quantitative Trait Loci (QTL) for soybean seed protein and sugar content can benefit plant breeders and the soybean market by accelerating the breeding process via marker-assisted selection. For this study, a population of recombinant inbred lines (RILs) was developed from a cross between R08-3221 (high protein and low sucrose) and R07-2000 (high sucrose and low protein). Phenotypic data for protein content were taken from the F2:4 and F2:5 generations. The DA7250 NIR analyzer and HPLC instruments were used to analyze total seed protein and sucrose content. Genotypic data were generated using analysis via the SoySNP6k chip. A total of four QTLs were identified in this study. Two QTLs for protein content were located on chromosomes 11 and 20, and two QTLs associated with sucrose content were located on chromosomes 14 and. 11, the latter of which co-localized with detected QTLs for protein, explaining 10% of the phenotypic variation for protein and sucrose content in soybean seed within the study population. Soybean breeding programs can use the results to improve soybean seed quality.

Development of energy-rich protein bars and in vitro determination of angiotensin I-converting enzyme inhibitory antihypertensive activities.

Three energy-rich protein (ERP) bars were prepared to meet the daily recommended dietary allowance (RDA) for the protein of Pakistani athletes. The bars were developed using dates, cheddar cheese (CC), whey protein isolate (WPI), roasted chickpea flour, and rice flour in different proportions. Bar #1 contained 64 g dates, 16 g dried apricots, 12 g WPI, and 8 g ripened CC. Bar #2 contained the same proportion of these ingredients with an addition of 12.5 g roasted chickpea flour, while bar #3 contained 6.25 g roasted rice and 6.25 g roasted chickpea flour. All the ingredients were homogeneously mixed into paste to form bars weighing 100-110 g per serving size. These bars were studied for the compositional analysis (moisture, protein, and lipid content), protein characterization through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and in vitro determination of the angiotensin I-converting enzyme (ACE-I) antihypertensive activity. Moisture and lipid content in bars were 22% and 0.057%-0.313%, respectively, while protein, fiber, and ash contents varied from 22.3% to 23.6%, 6.66 to 5.81, and 2.12% to 2.44%, respectively. The minimum energy content was recorded (272.70 Kcal/100 g) in bar #1 while bar #3 showed the highest energy content 274.65 Kcal/110 g with the addition of (5%) roasted chickpea and rice flour, respectively. Electrophoresis analysis of proteins in bar # 1 (cheese +WPI) showed the four bands at 62, 24, 20, and 12 kDa. Bar #2 (10% roasted chickpea flour) showed some additional bands at 40, 36, 34, and 28 kDa while relatively lower antihypertensive activity than bars #1 and 3. The study revealed that adding 10% roasted chickpea flour (bar #2) increased the protein content and diversity in proteins. It provided 40% proteins to athletes and could be helpful to meet their R.D.A. by consuming two bars/day.

Physicochemical properties of soy protein hydrolysate and its formulation and stability with encapsulated probiotic under in vitro gastrointestinal environment.

The objective of this study was to prepare protein isolate from defatted soybean and identify an optimal hydrolysis protocol to create improved hydrolysates and ascertain the optimum encapsulation technique for probiotics. Soy protein isolate (SPI) was prepared using an alkaline extraction procedure for solubility within a neutral, beverage-specific pH range. The soy protein hydrolysate (SPH) was prepared from aqueous extracted SPI using pepsin. The physicochemical properties of the SPH were investigated by solubility, degree of hydrolysis (DH), surface hydrophobicity, and electrophoresis. Hydrolysates from 2, 2.5, and 3 hr of hydrolysis time achieved the suitable DH between 2.5% to 5.0%. The 2.5 to 3 hr hydrolysates were also significantly more soluble than SPI at all pH levels from 85% to 95% solubility. Surface hydrophobicity of the hydrolysates ranged from 15 to 20 S0 values. Alginate (1%), resistant starch (2%), and probiotic culture (0.1%) were used as an encapsulation agent to protect probiotics. Alginate microcapsules were observed to be 1 mm in size using environmental scanning electron microscopy. The dried SPH and encapsulated probiotics with alginate in a dry powder formulation were tested for its gastrointestinal resistance and probiotic viability under in vitro simulated digestion. Approximately 1-log decrease was observed for all experimental groups after simulated digestion (final log colony forming units [CFU]/mL range: 6.55 to 6.19) with free probiotics having the lowest log CFU/mL (6.10 ± 0.10) value. No significant difference was observed among experimental groups for probiotic viability (P = 0.445). The findings of this research will provide an understanding of formulation for easily digestible protein and encapsulated probiotics. PRACTICAL APPLICATION: The findings of this research provide an understanding of improved formulation for more suitable soy protein hydrolysate and viability of encapsulated probiotics in gastrointestinal environment. Probiotics with the prebiotics in an encapsulated environment provide a technology for the enhancement of probiotics viability and for applications in suitable products for health and wellness.

Comparison of Cinnamon Essential Oils from Leaf and Bark with Respect to Antimicrobial Activity and Sensory Acceptability in Strawberry Shake.

Cinnamon leaf and bark essential oils have long been used as natural preservatives and flavoring agents in foods. This study determined antimicrobial effects of leaf and bark of cinnamon essential oils (CEOs) against 2 foodborne pathogens, Salmonella Typhimurium (S.T.) and Listeria monocytogenes (L.m.), at 2 initial bacterial levels (4- and 9-log CFU/mL) in strawberry shakes. The antimicrobial study of CEOs at 0.1% and 0.5% in strawberry shakes against S.T. and L.M. showed a significant difference (P < 0.05) in log reductions of both bacterial growth at low (4-log CFU/mL) and high (9-log CFU/mL) initial bacterial levels. Addition of 0.5% CEOs into strawberry shakes at 4 °C completely inhibited both bacteria after a period of 8 d storage. Shelf-life study showed that acidity and total solid content were not affected during storage. The strawberry shakes containing bark CEO had higher ratings of sensory acceptability compared to leaf CEO, with or without the addition of 1% masking agent. In conclusion, this study demonstrated that CEO derived from bark was better than that from leaf in terms of their antimicrobial activity and sensory aspect. PRACTICAL APPLICATION: This study demonstrates that essential oils derived from cinnamon bark and leaf have the potential to be used as natural antimicrobial ingredient in milk beverages with respect to sensory aspect. This finding promotes the acceptance of natural antimicrobials among consumers, while providing enhanced safer products to the food industry application.

Soybean peptide fractions inhibit human blood, breast and prostate cancer cell proliferation.

In this study, we examined in vitro the bio-activity of peptide fractions obtained from soybeans against blood (CCRF-CEM and Kasumi-3), breast (MCF-7), and prostate (PC-3) cancer cell proliferation. Gastro-intestinal treated peptide fractions (<5, 5-10 and 10-50 kDa) prepared from seed proteins of two high oleic acid soybean lines-N98-4445A, S03-543CR and one high protein line-R95-1705, were tested for anticancer activity against human breast, blood and prostate cancer cell lines. Anti-proliferative cell titer assay was conducted to assess the inhibitory effects of the peptide fractions, while trypan blue dye exclusion assay was used to determine the dose response of most effective fractions. Results showed that the peptide fractions inhibited the cancer cell lines up to 68.0% and the minimum concentration to get 50% inhibitory activity (IC50) ranged between 608 and 678 µg/mL. This multiple site in vitro cancer inhibition by GI friendly peptides could have the potential use as food ingredients or nutritional supplements in an alternative cancer therapy.

A Review: Supplementation of Foods with Essential Fatty Acids-Can It Turn a Breeze without Further Ado?

This paper focuses on the critical aspects of supplementation of foods with essential fatty acids (EFAs), the need, health benefits of supplementation and the constraints of the process. Current trend of supplementation of foods with EFAs has been gaining momentum and more research pioneers due to the health benefits in par with the direct intake of EFA supplements. Technologies including encapsulation, nanotechnology, molecular complexing, genetic engineering and more emerging means, hold promise to food supplementation with EFAs. Food trials with adoption of various technologies, studies of bioavailability and health benefits are still underway and crucial before EFA supplementation in foods can hit the market on a global scale.

Phenolic profile and antioxidant activity of extracts prepared from fermented heat-stabilized defatted rice bran.

Heat-stabilized, defatted rice bran (HDRB) serves as a potential source of phenolic compounds which have numerous purported health benefits. An estimated 70% of phenolics present in rice bran are esterified to the arabinoxylan residues of the cell walls. Release of such compounds could provide a value-added application for HDRB. The objective of this study was to extract and quantify phenolics from HDRB using fermentation technology. Out of 8 organisms selected for rice bran fermentation, Bacillus subtilis subspecies subtilis had the maximum phenolic release of 26.8 mg ferulic acid equivalents (FAE) per gram HDRB. Response surface methodology was used to further optimize the release of rice bran phenolics. An optimum of 28.6 mg FAE/g rice bran was predicted at 168 h, 0.01% inoculation level, and 100 mg HDRB/mL. Fermentation of HDRB for 96 h with B. subtilis subspecies subtilis resulted in a significant increase in phenolic yield, phenolic concentration, and radical scavenging capacity. Fermented rice bran had 4.86 mg gentistic acid, 1.38 mg caffeic acid, 6.03 mg syringic acid, 19.02 mg (-)-epicatechin, 4.08 mg p-courmaric acid, 4.64 mg ferulic acid, 10.04 mg sinapic acid, and 17.59 mg benzoic acid per 100 g fermented extract compared to 0.65 mg p-courmaric acid and 0.36 mg ferulic acid per 100 g nonfermented extract. The high phenolic content and antioxidant activity of fermented HDRB extract indicates that rice bran fermentation under optimized condition is a potential means of meeting the demand for an effective and affordable antioxidant.

Functional properties and Angiotensin-I converting enzyme inhibitory activity of soy-whey proteins and fractions.

Soy proteins when prepared to high purity can confer good functional properties and the whey by-product is a potential source for bioactivity. In this study, we determined the protein, moisture, fiber, solubility, foaming, emulsion properties, as well as Angiotensin-I converting enzyme (ACE-I) inhibitory activity of prepared soy-whey proteins and its fractions. The soy-whey proteins were fractionated into <5, >5, >10, and >50kDa using ultrafiltration. The expanded AACC methods were used to determine protein, moisture, and fiber analyses of the whey and its fractions. Solubility method was conducted to determine the protein solubility profile of the soy-whey and its fractions at varying pHs. Turbidimetric method was used to evaluate emulsifying activity (EA) and emulsion stability (ES). There were significant differences observed in moisture, protein and salt contents between unfractionated, >50kDa and smaller sized fractions. No significant differences were observed with phytic acid and total dietary fiber contents among all samples. The unfractionated whey protein and >50kDa fraction showed better solubility than other fractions. Unfractionated whey protein had the highest foam capacity (42.7mL) while the fraction >5kDa showed the greatest foaming stability (46min). The highest emulsion activity (0.33±0.1) and stability (825.1±45.1) was obtained with the >50kDa fraction while the unfractionated whey protein had the highest ACE-I inhibition activity. The findings indicate that soy-whey protein fraction (>50kDa) had good solubility, emulsion activity and stability, while the unfractionated whey protein exhibited the strongest ACE-I inhibition percentage (19%).

Physicochemical analysis of wheat flour fortified with vitamin A and three types of iron source and sensory analysis of bread using these flours.

BACKGROUND: Wheat flour is increasingly being fortified worldwide with vitamin A and iron. Research on high levels of fortification is limited; therefore, in this study, wheat flour was made under controlled conditions fortified with vitamin A at 30 000 or 70 000 retinol equivalents (RE) kg⁻¹ and three types of iron source at 66 mg kg⁻¹. RESULTS: Milling produced a uniform distribution of fortificants with no significant separation during packaging or transportation. Chemical and physical analyses demonstrated that the dual fortified flours had acceptable physicochemical properties of mixing tolerance, pasting curves, damaged starch and falling numbers. The level of vitamin A fortification compensated for initial loss caused during wheat processing. Overall, white breads baked from seven treatments of fortified flour had only 22% (eight out of 36) of the sensory attributes as being significantly different. However, the type of iron source may play a key role in modulating the sensory attributes of bread baked from the dual fortified flour with vitamin A and iron. CONCLUSION: The findings suggest that dual fortified flour with high or even lower levels of vitamin A and iron could be considered for food fortification programmes to reduce the prevalence of micronutrient undernutrition of vitamin A and iron in developing countries.

Electrostatic spraying of food-grade organic and inorganic acids and plant extracts to decontaminate Escherichia coli O157:H7 on spinach and iceberg lettuce.

UNLABELLED: The prevalence of foodborne illnesses is continually on rise. In the U.S.A., Escherichia coli O157:H7 (E. coli) has been associated with several outbreaks in minimally processed foods. Spinach and lettuce pose higher food safety risks and recurring food recalls suggest the insufficiency of current disinfection strategies. We aimed at offering a natural antimicrobial alternative using organic acids (malic, tartaric, and lactic acids [MA, TA, and LA, respectively]) and grape seed extract (GSE) and a novel application method using electrostatic spraying to evenly distribute the antimicrobials onto produce. Spinach and lettuce samples were washed, sanitized with sodium hypochlorite solution (6.25 mL/L), dip inoculated in water containing E. coli (7.0 log CFU/mL) for 24 h, and rewashed with sterile water to remove nonadhered pathogens. The samples were sprayed electrostatically with MA, LA, and GSE alone and in combinations and for comparison, with phosphoric acid (PA) and pH controls with deionized water adjusted to 1.5/2.3/3.6 and stored at 4 °C. When combined with LA (3%), MA (3%) showed 2.1 to 4.0 log CFU/g reduction of E. coli between the days 1 and 14 on spinach and 1.1 to 2.5 log CFU/g reduction on lettuce. Treatment with PA (1.5%) and PA (1.5%)-GSE (2%) exhibited 1.1 to 2.1 log CFU/g inhibition of E. coli on spinach during the 14-d storage. Our findings demonstrated the efficacy of electrostatic spraying of MA, LA, and GSE on fresh produce to improve the safety and lower the public health burden linked to produce contamination. PRACTICAL APPLICATION: Electrostatic spraying is an emerging technique that can be adopted to improve the distribution and application of antimicrobials during fresh produce sanitation. Relatively simple and quick, the process can access most/all parts of produce surface and offer protection from food pathogens. The use of malic and lactic acids with or without grape seed extract can serve as effective antimicrobials when sprayed electrostatically, lowering the risk from postcontamination issues with spinach and iceberg lettuce. This application technology can be extended to improve the commercial food safety of other produce, fruits, poultry, and meat.

Nutriproteomics: a promising tool to link diet and diseases in nutritional research.

Nutriproteomics is a nascent research arena, exploiting the dynamics of proteomic tools to characterize molecular and cellular changes in protein expression and function on a global level as well as judging the interaction of proteins with food nutrients. As nutrients are present in complex mixtures, the bioavailability and functions of each nutrient can be influenced by the presence of other nutrients/compounds and interactions. The first half of this review focuses on the techniques used as nutriproteomic tools for identification, quantification, characterization and analyses of proteins including, two-dimensional polyacrylamide electrophoresis, chromatography, mass spectrometry, microarray and other emerging technologies involving visual proteomics. The second half narrates the potential of nutriproteomics in medical and nutritional research for revolutionizing biomarker and drug development, nutraceutical discovery, biological process modeling, preclinical nutrition linking diet and diseases and structuring ways to a personalized nutrition. Though several challenges such as protein dynamics, analytical complexity, cost and resolution still exist, the scope of applying proteomics to nutrition is rapidly expanding and promising as more holistic strategies are emerging.

Shrimp shell peptide hydrolysates inhibit human cancer cell proliferation.

BACKGROUND: Shrimp wastes contain high-quality protein that is underutilized, and particularly peptides derived from shrimp wastes (normally used as animal feed) have not been utilized for bioactive properties. Hence the objective was to utilize shrimp waste proteins in generating peptides and to investigate these for cancer antiproliferative activities. The objectives involved hydrolyzing shrimp proteins (intact in shell) using a food-grade Cryotin enzyme, obtaining gastrointestinal resistant peptides, fractionation to generate < 10, 10-30 and > 30 kDa fractions, and evaluating for colon and liver cancer cell growth inhibitory effects. Three shrimp shells--whole langostino lobster shells from El Salvador (South America), shrimp shells from St Petersburg, FL (USA), and shrimp shell whites from the Gulf of Mexico, LA (USA)--were evaluated for the study. RESULTS: Peptide fractions (<10 and 10-30 kDa) obtained from shrimp shell whites (Gulf of Mexico) as well as from langostino shells (El Salvador) significantly inhibited the growth of both colon and liver cancer cells by 60%, while < 10 kDa fraction from shrimp shells (FL) inhibited growth of liver cancer cells alone by 55%, compared to controls. CONCLUSION: The promising anticancer peptide fractions from shrimp waste proteins has the potential for novel nutraceutical ingredient applications.

Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran.

Food-derived bioactive peptides promote functional activity against diseases and present as nutraceutical agents. The purpose of our research was to isolate and fully characterize peptide(s) derived from rice bran having anti-cancer properties. Gastrointestinal juices resistant peptide fractions were initially generated from heat stabilized de-fatted rice bran from which <5 kDa fraction was shown to inhibit proliferation of cancer cells. Based on these published findings the <5 kDa peptide fraction was selected for further characterization to obtain single pure peptide(s) with enhanced anti-cancer properties. Purification and characterization from the fraction was done employing chromatography and mass spectrometric techniques. Cancer cell viability was measured using a cell titer assay that uses a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; (MTS)] and the electron coupling reagent, phenazine methosulfate. Ion-exchange chromatography elutes that showed anti-cancer properties were further purified to liberate pure peptide. The pure peptide at 600-700 microg/mL dose caused 84% inhibition to colon cancer cells (Caco-2, HCT-116) growth, 80% to breast cancer cells (MCF-7, MDA-MB-231) growth and 84% to liver cancer cells (HepG-2) growth. Mass spectrometry analysis and de novo sequencing revealed the sequence of Glu-Gln-Arg-Pro-Arg for the peptide with a molecular mass of 685.378 Da. A novel pentapeptide was isolated from rice bran to possess cancer growth inhibitory properties on colon, breast, lung and liver cancer cells. This peptide could serve as a nutraceutical agent against cancer.

Electrostatic sprays of food-grade acids and plant extracts are more effective than conventional sprays in decontaminating Salmonella Typhimurium on spinach.

About 40000 people fall victim to Salmonella infections every year in the United States. Recent occurrences of Salmonella contaminated spinach and its recalls have accelerated the need for efficient antimicrobials targeting these pathogens. Our study was aimed at evaluating the inhibitory properties of malic, tartaric, and lactic acids, and grape seed extract (GSE) alone and in combinations and their application methods against Salmonella Typhimurium-inoculated spinach using a response surface method. Fresh spinach leaves were washed, disinfected with sodium hypochlorite solution (0.04% v/v), rewashed with sterile deionized (DI) water, and inoculated with a 2nd-day culture of S. Typhimurium (7.0 log CFU/mL). Adhered S. Typhimurium population on day 0 were 7.5 log CFU/g. These were treated with individual and combinations of organic acids with GSE or DI water (control) adjusted to the same pH as that of the test solutions with both the modes of application and leaves were refrigerated at 4 °C. Malic acid (2%) in combination with GSE (3%) or lactic acid (3%) sprayed electrostatically showed reductions of 2.6 to 3.3 log CFU/g compared to lower log reductions (0.0 to 0.3 log CFU/g) by day 14 if sprayed conventionally. These findings indicate that malic acid in combination with GSE/lactic acid solutions applied by electrostatic spraying exhibited higher inhibition of pathogens than conventional spraying and can be used for commercial applications to enhance food safety.

Effect of maturity stages and drying methods on the retention of selected nutrients and phytochemicals in bitter melon (Momordica charantia) leaf.

The purpose of this study was to investigate the nutrient and phytochemical composition of bitter melon leaves under varying maturity levels and drying techniques. Fresh, oven-dried, and freeze-dried leaves were evaluated over 3 maturity stages. In fresh leaves at various stages, crude fat, crude protein, and soluble dietary fiber contents ranged from 4.2% to 13.6%, 6.4% to 23.1%, and 0.04% to 3.50% on dry-weight basis, respectively. The contents of K, Ca, Mg, Fe, and Zn ranged from 1850.8 to 2811.8, 837.4 to 4978.2, 317.3 to 512.4, 8.4 to 16.7, and 4.1 to 5.9 mg/100 g dry-weight basis, respectively. Vitamin C, beta-carotene, and lutein contents ranged from 397.4 to 1275.1, 154.2 to 422.8, and 737.6 to 1304.6 microg/g dry-weight basis. The major flavonoids and phenolic acids were rutin, gentistic acid, and o-coumaric acid, which ranged from 7.57 to 12.75, 2.53 to 10.11, and 4.24 to 9.75 mg/g dry-weight basis, respectively. In oven-dried samples, 40.2% to 52.3% of vitamin C, 35.4% to 55.4% of beta-carotene, 25.6% to 71.6% of lutein, 26.4% to 84.0% of rutin, trace to 11.4% of gentistic acid, and 7.4% to 46.6% of o-coumaric acid were retained, while the retainment ratios of these components in freeze-dried samples were 84.7% to 99.0%, 76.4% to 99.3%, 90.4% to 96.1%, 39.8% to 99.3%, 24.1% to 68.4%, and 75.8% to 87.0%, respectively. The data showed that freeze-drying better preserves the nutrient and phytochemical quality of bitter melon leaves in comparison to oven-drying. Bitter melon leaf is a rich source of selected nutrients and phytochemicals.

Isoflavone contents in germinated soybean seeds.

The effect of germination on isoflavone contents in two soybean varieties (Hutcheson and Caviness) was investigated. Soybean seeds were soaked at 25 degrees C for 12 h, germinated at 40 degrees C, and freeze-dried. The isoflavone contents of dry, soaked, germinated (hypocotyl length at 0.5, 2.5, and 6.5 mm), and nongerminated seeds were determined by high performance liquid chromatography. The maximum amount of total isoflavone, genistein, and daidzein with their beta-glucoside conjugates was obtained when hypocotyl length of the germinated-seed from var. Hutcheson was 0.5 mm (2.491, 1.500, and 0.671 mg/g), and from var. Caviness was 2.5 mm (2.78, 1.523, and 0.905 mg/g). A dramatic increase in malonylgenistin and malonyldaidzin (1.305 mg/g and 0.476 mg/g in Hutcheson, and 1.308 mg/g and 0.677 mg/g in Caviness, respectively) was observed at these hypocotyl lengths. A decrease was observed after this stage. Genistein and daidzein contents were highest just after soaking. Glycitein and its beta-glucoside conjugates remained almost the same during germination. Controlled germination can be used to enhance isoflavone content in soybean seed.

Total phenolics and antioxidant activities of fenugreek, green tea, black tea, grape seed, ginger, rosemary, gotu kola, and ginkgo extracts, vitamin E, and tert-butylhydroquinone.

The total phenolics and antioxidant activities of fenugreek, green tea, black tea, grape seed, ginger, rosemary, gotu kola, and ginkgo extracts, vitamin E, and tert-butylhydroquinone, were determined. Grape seed and green tea were analyzed for their phenolic constituents using high-performance liquid chromatography. The total phenolics of the plant extracts, determined by the Folin-Ciocalteu method, ranged from 24.8 to 92.5 mg of chlorogenic acid equivalent/g dry material. The antioxidant activities of methanolic extracts determined by conjugated diene measurement of methyl linoleate were 3.4-86.3%. The antioxidant activity of the extracts using chicken fat by an oxidative stability instrument (4.6-10.2 h of induction time) followed a similar trend in antioxidant activity as determined by the Folin-Ciocalteu method. Seven phenolics in grape seed and green tea extracts were identified that ranged from 15.38 to 1158.49 and 18.3 to 1087.02 mg/100 g of extract, respectively. Plant extracts such as green tea and grape seed extracts can be used to retard lipid oxidation in a variety of food products.

Protein extraction from heat-stabilized defatted rice bran. 1. Physical processing and enzyme treatments.

Physical processing with or without enzyme treatments on protein extraction from heat-stabilized defatted rice bran (HDRB) was evaluated. Freeze-thaw, sonication, high-speed blending, and high-pressure methods extracted 12%, 15%, 16%, and 11% protein, respectively. Sonication (0-100%, 750 W), followed by amylase and combined amylase and protease treatments, extracted 25.6-33.9% and 54.0-57.8% protein, respectively. Blending followed by amylase and protease treatment extracted 5.0% more protein than the nonblended enzymatic treatments. High-pressure treatments, 0-800 MPa, with water or amylase-protease combinations, extracted 10.5-11.1% or 61.8-66.6% protein, respectively. These results suggest that physical processing in combination with enzyme treatments can be effective in extracting protein from HDRB.